Gel fractionator

ABSTRACT

A GEL FRACTIONATOR INCLUDES A CYLINDER FOR HOLDING A COLUMN OF GEL, TOGETHER WITH A PISTON ASSEMBLY FOR ADVANCING THE COLUMN THROUGH THE CYLINDER TOWARD A FRACTIONATING APPARATUS. THE PISTON ASSEMBLY INCLUDES AN AIR RELEASE VALVE OPERABLE FROM THE EXTERIOR OF THE CYLINDER FOR RELEASING TRAPPED AIR FROM ABOVE THE GEL COLUMN. THE FRACTIONATING ASSEMBLY INCLUDES A CUTTING SCREEN ENGAGED BY THE ADVANCING COLUMN FOR SEVERING THE ADVANCING COLUMN IN A GENERALLY AXIAL DIRECTION INTO SUBDIVISIONS OR SEGMENTS. A ROTATING WIRE BENEATH THE SCREEN SEVERS THE SUBDIVISIONS OF GEL AS THE GEL ADVANCES. THE WIRE IS MOUNTED ACROSS THE MOUTH OF A FUNNEL WHICH RECEIVES THE SEVERED GEL PARTICLES, AND THE FUNNEL INCLUDES STRUCTURE FOR DIRECTING A STREAM OF LIQUID AT THE UNDERSIDE OF THE SCREEN FOR FLUSHING THE SEVERED PARTICLES. THE FRACTIONATING OPERATION IS CARRIED OUT BY REPEATEDLY ADVANCING THE COLUMN A PREDETERMINED DISTANCE, PAUSING, AND CARRYING OUT SEVERAL DISCRETE FLUSHING OPERATIONS TO REMOVE ALL SEVERED PARTICLES.

May 23, 1972 w, E. en vsoN ET AL. 3,664,593

GEL-FRAcTIoNATon` Filed April L, 1970 Attorneys United States Patent3,664,593 GEL FRACTIONATOR Warren E. Gilson, 4801 Sheboygan Ave., andRobert E. Gilson, 4 Franklin Ave., both of Madison, Wis.

Filed Apr. 1, 1970, Ser. No. 24,640

Int. Cl. B02c 19/00 U.S. Cl. 241-60 10 Claims ABSTRACT OF THE DISCLOSUREA gel fractionator includes a cylinder for holding a column of gel,together with a piston assembly for advancing the column through thecylinder toward a fractionating apparatus. The piston assembly includesan air release valve operable from the exterior of the cylinder forreleasing trapped air from above the gel column. The fractionatingassembly includes a cutting screen engaged by the advancing column forsevering the advancing column in a generally axial direction intosubdivisions or segments. A rotating wire beneath the screen severs thesubdivisions of gel as the gel advances. The wire is mounted across themouth of a funnel which receives the severed gel particles, and thefunnel includes structure for directing a stream of liquid at theunderside of the screen for flushing the severed particles. Thefractionating operation is carried out by repeatedly advancing thecolumn a predetermined distance, pausing, and carrying out severaldiscrete dlushing operations to remove all severed particles.

The present invention relates to improvements in an apparatus and in amethod for fractionating a column of gel.

A gel fractionator is a device for dividing a cohesive column of gelmaterial into a series of individual samples or fractions. Such devicesare useful in connection with known analytical procedures wherein amaterial to be analyzed, such as for example a protein or othermacromolecular mixture, is dispersed by electrophoresis or the likewithin a column of a suitable gel material, such as a polyacrylamidegel;v The column is divided into sequential individual fractions duringthe fractionating process, and the fractions are individually analyzedas by determination of radio-activity in `a scintillation counter or thelike.

The traditional method used Ifor subdividing a gel column into fractionsis simple manual slicing with a knife or blade. This method yieldssatisfactory results in that very little zone distortion isintroduced-ie. the distribution f the sample along the column isaccurately reflected in the contents of the sequence of individualsamples. The diliiculty with manual slicing, of course, is that it isexcessively tedious and time consuming.

In order to overcome the necessity for manual slicing of the gel column,it has been proposed to provide a mechanical gel fractionator capableautomatically of dividing a gel column into fractions. U.S. Pat. No.3,451,629, Maizel describes a mechanical fractionator wherein a columnof gel is forced continuously through a restricted orice and carriedaway in a continuous liquid stream to be deposited in a series ofcontainers. Although representing an improvement in convenience overmanual methods, this device has been found to introduce an undesirableamount of zone distortion in the resulting fractions. It is believedthat this diticulty arises because the column of gel is reduced greatlyin cross section as the gel is Iforced or extruded through a restrictedorifice, resulting in undue distortion of the column.

Important objects of the present invention are to provide an improvedfractionating method and apparatus; to

ice

provide a gel fractionator overcoming the disadvantages of knowndevices; to provide a device and a method wherein zone distortion isovercome or minimized; and to provide an improved gel fractionatorhaving a novel air release arrangement.

In brief, a gel fractionator constructed in accordance with theprinciples of the present invention includes a cylinder for holding acolumn of gel and a piston assembly for advancing the gel in thecylinder toward a fractionating assembly including first cutting meansfor subdividing the column into axially oriented subdivisions or extensions and second cutting means for severing the subdivisions orextensions. During the cutting operations, the cross section of the gelcolumn is not substantially reduced. Means are provided for ushing thesevered gel particles from the region of the cutting means. Inaccordance with the novel method of the present invention the gel isadvanced a predetermined distance and then stopped during a delay periodin which the cutting oepration is completed. Following the delay period,several separate sequential iiush operations are carried out fully toWash the severed gel particles from the cutting region.

The present invention together with the above and other objects andadvantages may be better understood from consideration of the embodimentof the invention illustrated in the accompanying drawing wherein:

FIG. 1 is a fragmentary, broken, partly sectional and partlydiagrammatic view of a gel fractionator constructed in accordance withthe present invention; and

FIG. 2 is a greatly enlarged, fragmentary, partly sectional, explodedvieW of one portion of the apparatus of FIG. l.

Having reference now to the drawing, there is illustrated a gelfractionator designated as a whole by the reference numeral 104 andconstructed in accordance with the principles of the present invention.In general, the gel fractionator 10 includes a cylinder 12 held in arelatively fixed position by a support structure 14 and adapted tocontain a unitary, cohesive column of gel to be diwided into individualsamples or fractions. A relatively movable piston assembly generallydesignated as 16 is moved by a piston driving structure generallydesignated as 18 thereby to move the gel column within the cylinder 12.The piston assembly 16 includes a novel air release valve generallydesignated as 20 for permitting the release of air trapped between thecolumn and the piston assembly 16. The column of gel is moved by thepiston assembly 16 toward a novel fractionating assembly generallydesignated as 22 and serving to divide the advancing gel column intofractions which are discharged from an outlet 24.

AIf desired the fractionator 101 may be mounted directly on the carriagestructure of a fraction collector, in which case the fractions may bedeposited directly from the outlet 24 into a series of vials orcontainers (not shown) disposed successively beneath the outlet 24.Conveniently, the cylinder 12` comprises a glass cylinder, and ifdesired, may constitute the same cylinder in which the gel column andsample are subjected to a preceding electrophoresis operation. Thisarrangement avoids the necessity of transferring the column of gel froma different cylinder in order to carry out the fractionating operation.

The cylinder 12 is held in a relatively xed position by the supportstructure 14 including a lower holder 26 and an upper holder 28. -Lowerholder 26 includes a neck portion 30 loosely received in an opening 32in a lower mounting bracket 34, while the upper holder is threaded intoan opening 36 in an upper bracket 38. -In order to maintain the brackets34 and 38 rigidly in a parallel spaced arrangement corresponding to thelength of the cylinder 12, a support rod 40 is fixed to each of thebrackets 34 and 38.

In order to mount the cylinder 12 holding a column of gel and adispersed sample, the lower holder 26 is first positioned as illustratedon the lower bracket 34. The column 12 is then slipped through theopening 36 and into a recess 42 in the lower holder 26. The upper holder28 is then threaded into the opening 36, the upper end of the cylinderl12 being received in a recess 44. The upper holder 28 is threaded intothe upper bracket 38 until the cylinder 12 is held 'firmly in position.

After the cylinder 12 is mounted, the piston assembly 16 is inserted.The piston driving structure 18 is moved upwardly as viewed in FIG. 1 toits uppermost position to provide clearance for insertion of the pistonassembly 16 through an opening 46 in the upper holder 28 and into thetop of the cylinder 12.

In accordance with a feature of the present invention, the pistonassembly 16 includes the novel air release valve for releasing air thatmay be trapped in the cylinder 12 between the upper end of the gelcolumn and the lower end of the piston assembly. Referring morespecifically to the construction of the piston assembly 16, there isprovided an inner piston rod 48 received within an outer piston sleeve50. The air release valve 20 is made up of an enlarged piston headmember 52 of the rod 48, a resilient sealing gasket 54, and thelowermost end or edge 56 of the outer piston sleeve 50.

yOrdinarily the valve 20 assumes its illustrated position wherein thegasket 54 is held in sealing relation between the edge S6 and the headS2. Since the upper surface of the head 52 is sloped or cone-shaped, thegasket 54 is also forced outwardly into sealing relation against theinner wall of the cylinder 12. In this position, air and gel isprevented from leaking or extruding upwardly past the piston assembly16. lHowever, the release valve 20 can be opened from the exterior ofthe cylinder 12 in order to permit the release of trapped air. The upperend of the piston sleeve 50 is received in a recess 58 in a piston capelement 60, while the piston rodl 48 extends outwardly of the cap 60through a smaller diameter recess 62. A spring 64 held in compressionbetween the top of the cap 60 and the underside of a push button orenlargement 66 carried at the top of the rod 48 normally squeezes thegasket 54 between the edge 56 and the head 52.

When the piston assembly 16 is first inserted into the cylinder 12, airmay be trapped above the column of gel and below the piston head 52. Thepush button or enlargement 66 is pressed toward the cap 60` against theforce of the spring 64 to move the head 52 away from the edge 56 and tofree the gasket 54. In this position air can pass through a clearanceprovided between the head 52 and the wall of the cylinder 12 and aroundthe gasket 54. Air is then free to pass to atmosphere through theclearance between the piston sleeve 50 and the inner wall of thecylinder 12. When the head 52 engages the top of the gel column and thetrapped air is fully vented, the push button 66 is released and isreturned by the spring 64 to the position wherein the air release valve20 is fully closed by the force applied to the gasket 54 between theedge 56 and the head 52. The sealing force is increased when a drivingforce is applied to the piston sleeve 50 during the fractionatingprocess.

The piston driving structure 18 serves to force the piston assembly 16into the cylinder 12 thereby to move the gel column toward thefractionating assembly 22 and outlet 24. Although various types ofdriving structures may be used, in the illustrated arrangement there isprovided a drive plate 68 including a recess 70 receiving .the cap 60and including a shoulder 72 for applying a force by way of the cap 60 tothe piston sleeve 50. A reduced diameter recess 74 freely receives thepush button 66 and the spring 64.

In order to maintain the orientation of the drive plate 68 and to permitthe drive plate to be moved reciprocally relative to the supportstructure 14 and cylinder 12, the

4 plate is slidably mounted on a pair of parallel slide bars 76 and 78.

A driving force is applied to the drive plate 68 by means of a threadeddrive shaft 80 held against axial movement and extending through anopening 82 in the plate. The shaft 80 threadedly engages drive nut 84held on the plate 68 by means of a suitable holding bracket 86. Driveshaft 80 is rotated by means of a suitable driving connection indicatedby a broken line to a suitable drive motor 88 illustrated schematicallyin FIG. l. Rotation of the shaft 80 causes movement of the drive nut 84axially along the shaft and concurrent movement of the plate 68 alongthe slide bars 76 and 78. When the plate 68 is moved toward thesupporting structure 14, the piston assembly 16 is moved into thecylinder 12 to advance the gel column toward the fractionating assembly22- In accordance with an important aspect of the present invention thefractionating assembly 72 carries out a novel fractionating operation onthe column of gel advanced toward the assembly 22 by the piston assembly16 operating within the cylinder 12. Advantageously, the cross sectionalarea occupied by the advancing column of gel is not greatly restrictedin carrying out the fractionating operation and excessive extrusion ofthe gel and the resulting zone distortion are avoided. In accordancewith the invention, the advancing column of gel is first subdivided bysevering the gel in substantially axial directions to produce aplurality of generally axially oriented portions or extensions of thegel column. The subdivisions are then severed from the gel column bycutting in a substantially radial direction. The severed gel segmentsare ush'ed from the cutting area and are delivered through the outlet24.

Referring now more specifically to the structure of the fractionatingassembly 22, the lower holder 26 includes the recess 42 for receivingthe lowermost end of the cylinder 12, which recess 42 also defines ashoulder 90 upon which is mounted a first cutting structure 92 arrangedsubstantially normal -to the axis of the cylinder 12 and thussubstantially normal to the path of advancement of the gel column. Inthe illustrated arrangement the cutting member 92 comprises a screenwhich, for example, may be an 80 mesh screen. The screen 92 is heldagainst the shoulder 90 by means of a gasket 94 sandwiched between thescreen and the lower end of the cylinder 12.

Disposed directly beneath the screen 92 in a reduced diameter recess 96of the lower holder 26 is a gel severing and flushing unit generallydesignated as 98 and serving both to sever the gel subdivisions producedby advancing the gel through the screen 92 and to flush the severedsubdivisions from the region of the screen 92. As illustrated in greaterde-tail in FIG. 2, the unit 98 includes a body portion 100 mounted forrotation in the recess 96 together with a stem portion 102 extendingthrough a further recess 104 in the stern portion 30 in the lower holder26. The unit 98 includes an axially oriented central passageway 106communicating the outlet 24 with the region of the screen 92. Theuppermost end of the passageway 106 comprises a cone-shaped funnelpor-tion 107 having a mouth located beneath the screen 92 for receivingsevered portions of the gel column.

Supported across the mouth of the funnel portion of the unit 98 is asecond cutting means movable through a plane parallel to and just belowthe screen 92. The second cutting means comprises a wire 108, the endsof which are attached to the mouth of the funnel 107 as by inserting theends of the wire into notches 110 (FIG. 2).

Movement of the wire 108 is caused by rotating the gel severing andflushing unit 98. For this purpose, the stem portion 102 carries a drivegear 112 driven by a drive pinion 114 mounted on a rotatable shaft 116.As indicated by a broken line in FIG. 1, the shaft 116 is drivinglycoupled for rotation by a suitable drive motor 118. Operation of themotor 118 causes rotation of the stem 102 and thus of the unit 98 andaccordingly causes the wire 108 to rotate beneath the screen 92 andthereby carry out a cutting operation on the advancing gel subdivisions.In the illustrated arrangement, for example, the unit 98 is rotatedcontinuously throughout the fractionating operation at about twenty-liverevolutions per minute. Continuous rotation, however, is not necessaryto the method of the invention.

Since the subdivided and severed gel segments may tend to stick -to theadvancing `gel column and to the screen 102 and/or wire 108, thefractionating assembly 22 includes novel provision for flushing thesegments from the region of the screen 92. A suitable liquid pressurizedby a pump 119 ows through a conduit 120 to a passage 122 in the lowerholder 26 communicating with the recess 96. The outer peripheral wall ofthe body portion 100 of the unit 98 includes an annular recess 124 (FIG.2) communicating with passage 122. When pressurized lluid is channeledfrom the pump 119 to the recess 124, the fluid is directed by a passage126 extending through the body 100 from the recess 124 to the surface ofthe cone 107 and toward the underside of -the screen 92. The pressure ofthe uid and the diameter of the passage 126 are chosen so that the fluidsquirts upwardly against the screen. As the unit 98 is rotated, thefluid is directed over the entire cutting area `thereby efficiently andcompletely to carry away severed gel particles.

'In accordance with an important object of the invention thefractionation of gel with the apparatus is carried out in a novelsequence of steps assuring minimal zone distortion. In carrying out afractionating operation with a container located beneath the outlet 24,the first step is to advance the piston assembly through 16 through apredetermined increment of distance, which may for example be onemillimeter. This causes the gel column to be advanced into engagementwith the first cutting means, i.e. the screen 92. The screen 92 seversthe advancing gel in an axial direction to produce gel subdivisions orworm-like extensions of the gel column. After the gel has been advancedthrough an increment of distance, a pause of several seconds duration isprovided in order to allow the gel fully to ooze through the screen.During this pause, the unit 98 may be continuously rotating in orderthat the wire 108 severs the subdivided gel extensions. Alternatively,if desired, the unit 98 may be operated only brielly near the end of thepause.

In order to assure complete flushing, and prior to the next incrementaladvance of the piston assembly 16, a dlushing operation is carried out.Preferably several distinct flushing operations are provided byproviding a pulse of iluid through the passage 126, pausing briefly, andapplying a subsequent pulse of fluid. It has been found that three orfour distinct flushing operations during rotation of the unit 98 producevery complete removal of gel particles from the fractionating apparatus22. The particles and the liquid drop through the passage 106 and out ofthe outlet 24 and into the container or vial. Prior to repeating theprocess a next container is located beneath the vial.

Although the present invention has 'been described with reference to theillustrated embodiment, it should be understood that the details thereofdo not limit the invention as defined in the following claims.

What is claimed and desired to be secured by Letters Patent of theUnited States is:

1. A gel fractionator comprising a cylinder for holding a column of gelhaving a cross-sectional area, a piston means slidable within thecylinder for moving the column axially in the cylinder, first cuttingmeans positioned in the gel path and engaged by the advancing column forsubdividing the gel into a plurality of axially oriented sections havingsubstantially the same combined total cross-sectional area as thecross-sectional area of said column of gel, and second cutting meansmovable across the gel path for severing end portions from said axiallyoriented sections.

2. The gel fractionator of claim 1, said first cutting means comprisinga wire mesh screen arranged in a plane intersecting the gel path.

3. The gel fractionator of claim 1, said second cutting means comprisinga wire movable in a plane intersecting the gel path.

4. The gel fractionator of claim 3, said first cutting means comprisinga wire mesh screen arranged generally parallel to said plane ofmovement.

5. Gel fractionating apparatus comprising a cylinder for holding a gelcolumn; a screen disposed at one end of said cylinder, piston means forforcing the column through said screen thereby to subdivide the column,cutting means movable in a plane substantially parallel with said screenfor severing the gel column divisions, means lfor washing the severedsubdivisions from the screen, and means for collecting the severedsubdivisions.

6. The apparatus of claim 5, said collecting means comprising a funneldisposed beneath said screen.

7. The apparatus of claim 6, said cutting means comprising a wireextending across the mouth of said funnel, and means for rotating saidfunnel.

8. The apparatus of claim 7, said Washing means comprising an openingthrough the wall of said funnel directed at said screen.

9. A gel fractionator comprising a cylinder for holding a column of gel,a piston means slidable within the cylinder for moving the columnaxially in the cylinder, first cutting means positioned in the gel pathand engaged by the advancing column for subdividing the gel into aplurality of axially oriented sections, and second cutting means movableacross the gel path for severing end portions from said axially orientedsections, said piston means nicluding valve means for releasing air frombetween said piston means and the column, and 'valve operating meansdisposed externally of said cylinder.

10. A piston assembly for forcing a column of gel material through acylinder comprising an inner member including an enlarged head, an outersleeve surrounding said inner member, said head and sleeve havingdiameters smaller than the inside cylinder diameter, a resilient gasketsandwiched between said head and the end of said sleeve, resilient meansforcing said head and said sleeve toward one another and against saidgasket and forcing said gasket into se'aling relation against saidcylinder wall, and said inner member being movable against the force ofsaid resilient means to free said gasket and permit the release of airpast said head.

References Cited UNITED STATES PATENTS GRANVILLE Y. CUSTER, JR., PrimaryExaminer

